The present application includes a Sequence Listing filed on one (1) CD-R disc, provided in duplicate, containing a single file named pto_MDhMORF-8.txt, having 349 kilobytes, last modified on Apr. 3, 2001 and recorded Apr. 5, 2001. The Sequence Listing contained in said file on said disc is incorporated herein by reference in its entirety.
The present invention relates to novel isoforms of a human protein, and particularly relates to novel isoforms of human pregnancy-associated plasma protein-E.
Pregnancy-Associated Plasma Protein-A (PAPP-A) was first identified as a component of a circulating protein complex uniquely present in the serum of pregnant women. Principally of placental, that is, fetal, origin, and detectable in maternal serum as early as 4 weeks into gestation, PAPP-A has proven useful as a readily sampled marker for prenatal monitoring of fetal health and diagnosis of a number of human fetal abnormalities.
Maternal serum PAPP-A levels normally increase throughout gestation. Failure of PAPP-A levels to increase at the normal ratexe2x80x94that is, PAPP-A levels lower than the average for the respective gestational agexe2x80x94has been associated with a variety of fetal disorders.
For example, PAPP-A levels have been shown to be significantly lower than normal at 10-14 weeks gestation in pregnancies that subsequently result in miscarriage, pregnancy-induced hypertension, growth restriction, and pre-existing or gestational diabetes mellitus. Ong et al., Brit. J. Obstet. Gynaec. 107: 1265-70 (2000).
As another example, a statistical measure of maternal serum PAPP-A levels (the median multiple of the median (MoM)) is significantly decreased (less than the 5th centile of normal in 78% of cases) during the first trimester in cases of fetal trisomy 18. Tul et al., Prenat. Diagn. 19: 1035-42 (1999). When measurement of PAPP-A is combined with measurement of maternal serum free xcex2hCG, fetal nuchal translucency, and maternal age, 89% of cases of trisomy 18 can be detected with a 1% false-positive rate.
In the second trimester of pregnancy, maternal serum levels of PAPP-A are reduced more markedly than either alpha fetoprotein (xcex1FP) or free beta-hCG in cases of trisomy 18; one study reports levels of PAPP-A lower than the 5% centile of normal in 93% of the cases. Spencer et al., Prenat. Diagn. 19: 1127-34 (1999).
Median maternal serum levels of PAPP-A are also significantly reduced at 8 to 14 weeks in trisomy 21 gestations. Screening using maternal age, serum-free xcex2-hCG, and PAPP-A at 10 weeks of pregnancy has been demonstrated to provide better prediction of fetal trisomy 21 than one standard test (levels of alpha-fetoprotein and hCG, in conduction with maternal age), and equal predictive value to the test of xcex1FP, unconjugated estriol, hCG, and maternal age at 15-22 weeks. Wald et al., Br. J. Obstet. Gynaecol. 103(5):407-412 (1996).
Although first discovered based upon its primary expression in placental tissue, PAPP-A has also been detected in ovaries, with expression restricted to healthy antral follicles in granulosa cells and healthy corpora lutea (CL) in a subset of large luteal cells, a pattern of expression consistent with a role for PAPP-A at the very outset of pregnancy, through control of survival, growth, and/or differentiation of the dominant ovarian follicle. Hourvitz et al., J. Clin. Endocrinol. Metab. 85:4916-4920 (2000).
PAPP-A is a member of the metzincin superfamily of metalloproteinases.
The metzincin gene superfamily was first identified based upon topological and sequence relationships among the astacins, adamalysins, serralysins, and matrix metalloproteinases. These zinc endopeptidases share topological similarity with respect to a five-stranded beta-sheet and three alpha-helices arranged in typical sequential order. The common consensus motif, HEXXHXXGXXH, found in PAPP-A at residues 482-492, contains three histidine residues which are involved in binding of the catalytically essential zinc ion. Stocker et. al., Protein Sci. 4:823-40 (1995). Metzincins also possess a conserved methionine residue in spaced relationship to the zinc-binding motif; in PAPP-A, the conserved methionine is believed to be the methionine at residue 556.
PAPP-A has been demonstrated specifically to cleave insulin-like growth factor binding protein 4 (IGFBP-4), Lawrence et al., Proc. Natl. Acad. Sci. USA 96:3149-3153 (1999), and to be the dominating, if not sole, IGFBP-4 protease present in the circulation, Overgaard et al., J. Biol. Chem. 275:41128-31133 (2000). IGFBP-4 is one of six known inhibitors of IGF action in vitro; like other IGFBPs, cleavage of IGFBP-4 has been shown to abolish its ability to inhibit IGF activity. The cleavage specificity of PAPP-A for IGFBP-4 implicates PAPP-A in normal and pathological physiology of insulin-like growth factor (IGF). Overgaard et al., J. Biol. Chem. 275: 31128-33 (2000).
PAPP-A exists in pregnancy serum as a covalent, heterotetrameric 2:2 complex with the proform of eosinophil major basic protein (proMBP); pro-MBP appears to inhibit PAPP-A""s protease activity. Overgaard et al., J. Biol. Chem. 275:31128-33 (2000). Conversely, IGF appears to be a necessary cofactor or agonist for PAPP-A protease activity, leading to a feedback network controlling IGF availability.
Reports in the literature of pregnancy-related proteins related in sequence to PAPP-A, termed PAPP-B, PAPP-C, and PAPP-D, have proven spurious. Farr et al., Biochim. Biophys. Acta 1493:356-362 (2000) recently identified a cDNA that encodes a protein related in primary sequence and protein domain structure to PAPP-A and that is expressed primarily in placenta, which they term PAPP-E. FARR et al. report that the cDNA encodes a complete open reading frame.
Recent reports suggest that at least one-third, and likely a higher percentage, of human genes are alternatively spliced. Hanke et al., Trends Genet. 15(1):389-390 (1999); Mironov et al., Genome Res. 9:1288-93 (1999); Brett et al., FEBS Lett. 474(1):83-6 (2000). Alternative splicing has been proposed to account for at least part of the difference between the number of genes recently called from the completed human genome draft sequencexe2x80x9430,000 to 40,000 (Genome International Sequencing Consortium, Nature 409:860-921 (Feb. 15 2001)xe2x80x94and earlier predictions of human gene number that routinely ranged as high as 120,000, Liang et al., Nature Genet. 25(2):239-240 (2000). With the Drosophila homolog of one human gene reported to have 38,000 potential alternatively spliced variants, Schmucker et al., Cell 101:671 (2000), it now appears that alternative splicing may permit the relatively small number of human coding regions to encode millions, perhaps tens of millions, of structurally distinct proteins and protein isoforms.
With increasing age, women experience decrease in ovarian reserve and, upon conception, an increased incidence of aneuploid gestations. Given a likely role of PAPP-A in controlling ovarian follicular maturation, and its proven clinical utility as a predictor of fetal abnormality during gestation, PAPP-A has potential therapeutic as well as diagnostic roles in clinical infertility practice.
With the recent identification of a protein that is related to the clinically useful prenatal diagnostic marker, human PAPP-A, and the recognition that alternatively spliced isoforms of proteins are as critical to metabolic and physiologic function as proteins that are separately encoded, there is a need to identify and to characterize additional isoforms of the PAPP-E protein.
The present invention solves these and other needs in the art by providing isolated nucleic acids that encode three novel isoforms of hPAPP-E, and fragments thereof.
In other aspects, the invention provides vectors for propagating and expressing the nucleic acids of the present invention, host cells comprising the nucleic acids and vectors of the present invention, proteins, protein fragments, and protein fusions of the novel PAPP-E isoforms, and antibodies thereto.
The invention further provides pharmaceutical formulations of the nucleic acids, proteins, and antibodies of the present invention.
In other aspects, the invention provides transgenic cells and non-human organisms comprising human PAPP-E isoform nucleic acids, and transgenic cells and non-human organisms with targeted disruption of the endogenous orthologue of the human PAPP-E gene.
The invention additionally provides diagnostic, investigational, and therapeutic methods based on the PAPP-E nucleic acids, proteins, and antibodies of the present invention.